Experimental overview A sequential protein and peptide IMAC enrichment was analyzed by four different LC-MS/MS strategies. Experimental overview A sequential.

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binding sites 58 of the 473 unambiguously assigned phosphorylation sites are predicted by Scansite to be sites for binding. 50 of these correspond.

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A, high resolution MS/MS spectrum (lower panel) of 1435

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Complementary identification and novel protein discovery

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Experimental overview A sequential protein and peptide IMAC enrichment was analyzed by four different LC-MS/MS strategies. Experimental overview A sequential protein and peptide IMAC enrichment was analyzed by four different LC-MS/MS strategies. Three DDAs with varying LC gradient times were performed (experiments A, B, and C) termed “Multiple DDA.” Enzymatic dephosphorylation (dephos) of the double IMAC was performed and analyzed using the same LC-MS/MS conditions as those used in experiment C (experiment D). Two iterative DDA strategies were also used: in experiment E, an exclusion (excl) list was generated and used as a filter for subsequent DDA experiments, and in experiment F, and EMRT inclusion (incl) list was generated using Protein Expression (MSE) analysis and was used for subsequent DDA analyses. *, eight of 69 proteins were still phosphorylated, and 18 of 145 peptides were still phosphorylated; the remaining 127 previously phosphorylated peptides are described in supplemental Table 5. NR, non-redundant; seq, sequences. Mark O. Collins et al. Mol Cell Proteomics 2008;7:1331-1348 © 2008 The American Society for Biochemistry and Molecular Biology